The rise in antibiotic resistance among bacterial pathogens has rekindled the threat of infectious disease and accompanying morbidity and mortality. Thus, it is imperative that we develop new strategies to combat bacterial infections. Further, new understanding of the beneficial roles of normal host microbiota in human nutrition, immunology and health suggests that targeted therapies that eliminate harmful bacteria from sites of infection without affecting beneficial microbes are needed. One promising strategy for developing such treatments is to intervene in the mechanisms by which pathogens colonize the particular niches in which they cause infection. An important mechanism affecting the tropisms of a wide range of Gram-negative bacteria is the elaboration of adhesive extracellular fibers called chaperone-usher pathway (CUP) pili. One of the best studied of these systems is type 1 pili and its mannose binding adhesin, FimH, which is critical for host pathogen interactions throughout the course of urinary tract infections (UTIs). UTI: i) is one of the most common bacterial infections; ii) primarily affects otherwise healthy females (50% of women will have a UTI) and; iii) is highly recurrent and is associated with significant morbidity and economic impact, with over $2.5 billion spent annually on treatment. Uropathogenic E. coli (UPEC), the causative agent of ~80% of UTIs, is becoming increasingly antibiotic-resistant. Structural, genetic and biophysical studies have defined the binding interactions between mannose and FimH and indicate that conformational equilibria within FimH regulate its function and role in UTI pathogenesis. Surprisingly, little to nothing is known regarding the structure, function and mechanism of action of the majority of common UPEC CUP adhesins. Therefore, this proposal will elucidate the structures and functions of the two-domain CUP adhesins from two prototypical UPEC strains, used widely to study UTIs: i) UTI89 (a human clinical cystitis isolate) and ii) CFT073 (a human clinical pyelonephritis/urosepsis isolate. Each of these strains encodes complete CUP pilus assembly operons for type 1, Yqi, Yfc1, P, F1C/S, Yeh, Yad3, and Mat pili. In addition, UTI89 encodes a complete F17-like CUP operon and CFT073 encodes complete F9 (Fml) and Auf CUP operons. This proposal will study how conformational equilibria of the FimH adhesin and the FimA rod of the type 1 pilus impact host-pathogen interactions critical in pathogenesis (Aim 1) and determine the function of common UPEC two-domain CUP adhesins, determining UPEC adhesin tissue tropisms, ligand specificity, structure-function correlates and conformational dynamics (Aim 2). This proposal will also investigate structure/function correlates within the family of FimH-like adhesins, revealing likely evolutionary steps leading from one binding specificity to another within the family of FimH-like adhesins and characterizing the probable influence of host innate immunity on natural selection in adhesins (Aim 3). The successful completion of this proposal will give a holistic view into the functional CUP piliome of UPEC, providing new insights into the dynamic function of pili and development of novel therapeutics.